N-acetylcysteamine thioester.6 In the study to confirm the
hypothetical tropinone binding mode, Nakajima et al.
substituted five substrate-binding residues of one tropinone
reductase (TR-1) with those found in the corresponding
positions of the other tropinone reductase (TR-2) in the
biosynthetic pathway of tropane alkaloids, resulting in the
switch of stereospecificity of TR-1 into that of TR-2 and
vice versa.7 Herein, we report the first example of enzyme-
substrate docking-guided point mutation of the substrate-
binding residues in a carbonyl reductase, generating mutant
enzymes with reversed enantiopreference and enhanced
enantioselectivity toward the reduction of para-substituted
acetophenones. These results provide some insight into the
fundamental question of how substrate-binding residues
affect substrate binding orientation and thus control the
reaction stereoselectivity.
(S)-enantiomer of product alcohol (Figure 1A) or (R)-
counterpart (Figure 1B), respectively. From these docking
studies, which are qualitatively consistent with the low
observed enantioselectivity, it was also seen that residues
Q245 and M242 are in close proximity to the para substituent
of the acetophenones in both conformations. We reasoned
that such close interaction might be responsible for the
observed low enantioselectivity in the SSCR-catalyzed
reduction of para-substituted acetophenones, and that muta-
tion of the residues Q245 and M242 in the catalytic site might
improve the enzyme enantioselectivity, with hydrogen bond-
ing or hydrophobic interactions playing significant roles. In
the present study, the residue Q245 was mutated to all other
19 amino acids, and the mutants were screened for enhanced
enantioselectivity toward the reduction of para-substituted
acetophenones.
Although the carbonyl reductase from Sporobolomyces
salmonicolor (SSCR) catalyzes the reduction of various
ketones to the corresponding chiral alcohols in excellent
enantiomeric purity, it shows low enantioselectivity for the
reduction of para-substituted acetophenones (14-59% ee).8
To better understand the enantioselective versatility in this
ketone reduction, an initial substrate-enzyme docking study
of 4′-methoxyacetophenone into the crystal structure of
SSCR9 was performed using ICM-Pro 3.4.9d.10 During these
simulations, two opposite conformations which are energeti-
cally close to each other have been found in the high scoring
docking conformations. Figure 1 shows the opposite con-
formations of 4′-methoxyacetophenone, which yield the
A focused library of mutants was created by saturation
mutagenesis of the residue Q245 in the catalytic cavity of
the carbonyl reductase from S. salmonicolor. The resulting
mutant library was screened using 4′-methoxyacetophenone
as substrate. The colonies which showed higher activity than
wild-type SSCR enzyme were selected to further determine
their enantioselectivity. Surprisingly, five colonies were
found to catalyze the reduction of 4′-methoxyacetophenone
to (S)-1-(4′-methoxyphenyl)ethanol with ee values of
79-98%, while (R)-1-(4′-methoxyphenyl)ethanol was ob-
tained in 57% ee with the wild-type SSCR enzyme. No
colony showing higher activity than the wild-type SSCR
enzyme was found to catalyze the reduction of 4′-methoxy-
acetophenone to (R)-1-(4′-methoxyphenyl)ethanol. Sequenc-
ing of the five colonies revealed three colonies showing the
same mutation, Q245L, while the other two colonies showed
mutations Q245H and Q245P, respectively.
(3) (a) Cyclopentanone monooxygenase: Clouthier, C. M.; Kayser, M.
M.; Reetz, M. T. J. Org. Chem. 2006, 71, 8431. (b) Cyclohexanone
monooxygenase: Reetz, M. T.; Brunner, B.; Schneider, T.; Schulz, F.;
Clouthier, C. M.; Kayser, M. M. Angew. Chem., Int. Ed. 2004, 43, 4075.
(c) Epoxide hydrolase, Reetz, M. T.; Wang, L. W.; Bocola, M. Angew.
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J.; Sonke, T.; Wubbolts, M. G.; Janssen, D. B. Chem. Biol. 2004, 11, 981.
(d) Hydroxynitrile lyase, Weis, R.; Gaisberger, R.; Skranc, W.; Gruber,
K.; Glieder, A. Angew. Chem., Int. Ed. 2005, 44, 4700. (e) Aldolase,
Williams, G. J.; Domann, S.; Nelson, A.; Berry, A. Proc. Natl. Acad. Sci.
U.S.A. 2003, 100, 3143. (f) Vanillyl-alcohol oxidase: van den Heuvel, R.
H. H.; Fraaije, M. W.; Ferrer, M.; Mattevi, A.; van Berkel, W. J. H. Proc.
Natl. Acad. Sci. U.S.A. 2000, 97, 9455. (g) Hydantoinase, May, O.; Nguyen,
P. T.; Arnold, F. H. Nature Biotechnol. 2000, 18, 317. (h) Phosphotri-
esterase: Chen-Goodspeed, M.; Sogorb, M. A.; Wu, F.; Raushel, F. M.
Biochemistry 2001, 40, 1332. (i) Arylmalonate decarboxylase: Ijima, Y.;
Matoishi, K.; Terao, Y.; Doi, N.; Yanagawa, H.; Ohta, H. Chem. Commun.
2005, 877.
These mutant SSCR enzymes were further screened to
determine whether they also inverted enantioselectivity
toward the reduction of other para-substituted acetophenones.
The results as summarized in Table 1 show that when the
residue Q245 was replaced with H, P, or L (Q245H, Q245P
and Q245L), reductions of all other para-substituted aceto-
phenones gave (S)-configurated chiral alcohols in greater than
90% ee, while the unsubstituted acetophenone was reduced
in a relatively lower enantioselectivity. When compared to
the wild-type SSCR, which catalyzed the reduction to give
(R)-enantiomer in 14-59% ee, these mutant SSCR enzymes
exhibited inverted enantiopreference and enhanced enantio-
selectivity. Therefore, the residue 245 in the catalytic cavity
plays an important role in determining the enantioselectivity
for the reduction of the para-substituted acetophenones.
Furthermore, this residue affected the enzyme activity for
the reduction of acetophenones. For example, compared to
the wild-type SSCR and mutant Q245L, mutants Q245H and
Q245P greatly improved the enzyme activity toward the
reduction of acetophenones when the para substituent was
Cl or Br.
(4) Li, Z.; Butikofer, L.; Witholt, B. Angew. Chem.,Int. Ed. 2004, 43,
1698.
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Chem. Biol. 2006, 13, 277. (b) O’Hare, H. M.; Baerga-Ortiz, A.; Popovic,
B.; Spencer, J. B.; Leadlay, P. F. Chem. Biol. 2006, 13, 287.
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To gain insight into how the single mutation at residue
245 results in such a drastic change of enantioselectivity for
the reduction of para-substituted acetophenones, in silico
mutagenesis of Q245 to H and docking of a variety of para-
526
Org. Lett., Vol. 10, No. 4, 2008